Analysis of the complete genome sequences of Clostridium perfringens strains harbouring the binary enterotoxin BEC gene and comparative genomics of pCP13-like family plasmids

Background BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains. Results In this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1–3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their ‘accessory regions’ can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains. Conclusion This study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-022-08453-4.


Introduction
Clostridium perfringens is a gram-positive, spore-forming bacterium that causes a variety of diseases in humans and animals. The pathogenicity of C. perfringens is mainly attributed to the toxins produced by the bacterium, and more than 20 types of toxins have been reported [1][2][3][4][5][6]. Each C. perfringens strain produces a distinct but limited number of these toxins, and strains were historically classified into five toxinotypes according to their ability to express four types of toxins: α-toxin (phospholipase C; PLC; plc), β-toxin (CPB; cpb), ε-toxin (ETX; etx), and ι-toxin (ITX; iap/ibp) [1]. This typing scheme was recently updated by adding two more toxins, C. perfringens enterotoxin (CPE; cpe) and necrotic enteritis toxin B (NetB; netB), which cause human gastrointestinal (GI) disease and necrotic enteritis in poultry, respectively, resulting in a new classification with seven toxinotypes (types A-G) [7][8][9][10].
Two of the six typing toxin genes are located on the chromosome; plc, which is carried by all C. perfringens strains, is always located on the chromosome, while cpe is both on the chromosome (chromosomal cpe) and on a plasmid (plasmid-borne cpe). The other toxin genes, cpb, etx, iap/ibp, and netB, are located on plasmids [11,12]. In addition to these plasmid-encoded toxins, it was recently shown that C. perfringens strains causing GI diseases harbour virulence plasmids encoding novel toxin genes. For example, recently identified C. perfringens toxin genes, such as tpeL (encoding the large cytotoxin TpeL) reported in 2007; netB reported in 2008; and three toxin genes, netE, netF, and netG, reported in 2015 were all found to reside on conjugative plasmids [13][14][15]. This suggests that conjugative plasmids play an important role in disseminating virulence genes in this bacterium via horizontal gene transfer (HGT), which further increases the toxin repertoire of C. perfringens [16]. In 2014, we also reported a novel plasmid-encoded enterotoxin, binary enterotoxin of C. perfringens (BEC/CPILE), in strains that caused food poisoning in two outbreaks in Osaka and Tochigi Prefectures in Japan in 2009 and 2010, respectively [17][18][19]. BEC consists of an enzymatic component (BECa; becA) and a cell-binding component (BECb; becB) and is homologous to the binary ADP-ribosylating toxin, ι-toxin, which causes gastroenteritis in cattle and rabbits and is encoded by a conjugative plasmid present in type E strains. In contrast, the two isolates, strain OS1 from the Osaka cases and TS1 from the Tochigi cases, that caused these outbreaks were classified as type A strains [17]. Since most reported type A strains do not possess any toxin-encoding plasmids, they were not known to cause GI diseases in humans until bec-harbouring strains were reported [16]. This suggests that these toxin genes may be acquired via HGT.
Two conjugative plasmid families of C. perfringens, pCW3 and pCP13, have been reported. The archetypical plasmid of the pCW3 family, pCW3, has a conjugation region that is designated as the 'transfer of clostridial plasmid' (tcp) locus [20]. This plasmid is also a carrier of clinically relevant toxins and antimicrobial resistance genes. These genes are encoded in the accessory or variable region, which integrates multiple foreign genes [20,21]. The archetypical plasmid of the pCP13 family, pCP13, was initially suspected to be non-conjugative because of the absence of any conjugation loci [22]. Although the dissemination of this plasmid family in natural environments is unknown, it has been reported that pCP13 is conjugative, with an extremely high transfer frequency of ~10 −1 transconjugants/donor cells, which is comparable to that of a pCW3-like family plasmid [22]. In this study, a locus encoding conjugation machinery that is homologous to the type 4 secretion system (T4SS) and was named the 'pCP13 C. perfringens' (Pcp) locus was newly defined [22]. Unlike pCW3-like family plasmids, no toxin or antimicrobial resistance genes have been identified in the accessory region of pCP13like family plasmids, except for a consensus variant of the β-toxin2 gene (CPB2; cpb2) that is associated with intestinal diseases in horses and piglets [23]. Therefore, it is noteworthy that the recent sequencing of plasmids in OS1 and TS1 strains revealed that becA/becB genes, which have been associated with human GI diseases, are located in the accessory regions of ~54 kbp plasmids containing almost identical Pcp loci (98% identity) to that of pCP13 [17,22].
Since the first report of BEC by us [17], three other groups have detected bec-harbouring C. perfringens strains, including strain W5052, isolated from the faeces of patients with gastroenteritis and food in Tokyo as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains.
Prefecture, Japan (in October 1997 and June 2003, respectively) [19,24]; strain CP653, isolated from the faeces of an outpatient in Hokkaido Prefecture, Japan (June 2019) [25]; and strain IQ3 (alternatively named Q135.2), isolated from the faeces of a healthy child in London, the UK (September 2019) [26]. We also detected three more bec-harbouring C. perfringens strains, O13-19, A18-256, and A19-1, in Japan. Strain O13-19 was isolated from the faeces of a patient with gastroenteritis due to a non-C. perfringens foodborne illness in Osaka Prefecture (June 2013). The other two strains, A18-256 and A19-1, were individually isolated from the faeces of patients with foodborne gastroenteritis caused by bec-harbouring C. perfringens in October 2018 and from oysters purchased at a local market in January 2019, respectively, both in Aichi Prefecture, Japan. Therefore, at least eight isolates with different geographical and temporal origins and variation in the symptomatology of the host have been identified ( Table S1). As of 19 December 2020, a total of 211 assembled genomes of C. perfringens were deposited in the public database; however, only one (Q135.2) was a bec-harbouring strain. Thus, to understand the genomic epidemiology of bec-harbouring C. perfringens strains, high-throughput sequencing of other bec-harbouring C. perfringens strains is required.
In this study, we sequenced the genomes of five becharbouring strains, OS1, TS1, O13-19, A18-256, and A19-1, using a hybrid approach [17]. The obtained genomic information showed that the bec genes are encoded on the pCP13-like family plasmids, most of which could have been acquired via HGT events mediated by the Pcp conjugation locus. Comparative genomics of these newly determined five C. perfringens genomes along with the 211 assembled genomes in the public database showed that the pCP13-like family plasmids are unexpectedly widespread and are distributed across several C. perfringens phylogenetic clades. These findings not only illuminate the mechanisms underlying the dissemination of bec-harbouring strains but also shed new light on pCP13-like family plasmids to improve our understanding of C. perfringens virulence.

Results and Discussion
Complete genome sequencing of bec-harbouring C. perfringens strains Five bec-harbouring C. perfringens isolates, OS1, TS1, O13-19, A18-256, and A19-1, were sequenced using short-and long-read sequencing platforms. This hybrid sequencing approach enabled the raw reads to be assembled into a complete genome. The coverage of the short reads was >45× and that of the long reads was >140×, which is sufficient to obtain complete genome sequences with high accuracy (Tables 1 and S2-1-3). These genomes were confirmed to be C. perfringens at the species level based on the average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values of ≥97.1% and ≥ 74.1%, respectively (Table 1). These genomes had 28.30-28.43% GC content and contained an average of 3023 CDS, 96 tRNA genes, and 31 rRNA genes ( Table 1).

Exploring the clostridial toxin and antimicrobial resistance genes, CRISPR regions, and prophage regions in the C. perfringens strains
We explored the clostridial toxin genes, antimicrobial resistance genes, CRISPR regions, and prophage regions in the five complete genomes of the sequenced bec-harbouring strains. Of the six typing genes, plc was solely detected on the chromosome, and five strains were therefore classified as type A [27]. The non-typing toxin genes, collagenase gene (colA), perfringolysin O gene (pfoA), alpha-clostripain gene (cloSI), five hyaluronidase genes (nagH, nagI, nagJ, nagK, and nagL), and three sialidase genes (nanH, nanI, and nanJ), were shared by all five strains and were located on their chromosomes ( Fig. 2 and Table S3). Four strains, excluding OS1, had an alveolysin (alv) gene on their chromosome. This toxin, belonging to a family of thiol-activated cytolysins, is frequently found in Bacillus alvei [28], but is less common in C. perfringens strains [29]. It is interesting to note that no known virulence genes were found on either of the plasmids in the bec-harbouring strains, except the becA/B genes ( Fig. 2 and Table S3).

Phylogenetic analysis of C. perfringens
To elucidate the phylogeny of the bec-harbouring strains, we constructed a maximum likelihood phylogenetic tree based on the core genome of 216 C. perfringens strains, including the five newly determined complete genomes and others extracted from GenBank (Fig. 2). The toxinotype and the presence or absence of typical C. perfringens toxin genes in each strain are shown in Fig. 2. The phylogenetic tree suggests that C. perfringens strains are divided into five major clades, as was reported previously by Feng et al. [31]. Clade 4 consists of a large number of strains that can be further divided into several clades based on hierarchical Bayesian clustering by RhierBAPS, as was also pointed out by Feng et al. [31,32] (Fig. S2 and Table S4). In the present study, we combined these clades into one large clade to eliminate complexity (Fig. 2).
Of the six bec-harbouring isolates, including strain Q135.2 from the UK [26], five (Q135.2, TS1, O13-19, A18-256, and A19-1) were clustered in clade 3 (Fig. 2). Clade 3 mainly consisted of a large collection of 'JFP' strains, which were isolated from a North American dog and in a study of horse C. perfringens haemorrhagic enteritis [33]. These strains were categorised as either type F or G, and they possess plasmid-borne cpe or netB and plc, respectively. Among the four currently known type E strains, three strains (cp508.17, cp515.17, and Q061.2) were also clustered in this clade. Given that the five bec-harbouring strains were categorised as toxinotype A, the phylogenetic analysis showed that clade 3 contained strains, most of which possessed plasmidborne toxin genes, with relatively diverse toxinotypes (A, E, F, and G; Fig. 2).
While most of the bec-harbouring strains belonged to clade 3, strain OS1 was classified into clade 4 along with strain 13 carrying pCP13 [22], which encodes a conjugative Pcp locus and has a 38 kbp region with sequence similarity to bec-encoding plasmids [17,22]. Considering that the bec-encoding plasmids carry a conjugative Pcp locus, we hypothesised that the distribution of bec genes across different C. perfringens clades was caused by an inter-clade HGT event.

Detection of HGT events among six bec-harbouring strains based on analysis of InDels and SNPs
We compared six bec-encoding plasmids, including the plasmids sequenced in this study and one (pIQ3b) from strain Q135.2 [26] (Fig. 1), which showed that the sequence similarity among these six bec-encoding plasmids was exceptionally high (>99.81%). The plasmid sequences were then aligned using parA as a starting point, and slight differences in SNPs and InDels were confirmed (Tables 3 and 4). These differences consequently divided these plasmids into two groups with distinct sequence lengths: group A (54,478 bp; TS1 and A18-256) and group B (54,536 bp; OS1, O13-19, A19-1, and Q135.  (Table 3). In each group, several minor differences in SNPs were detected, at position 45,011 in group A and at positions 12,297, 28,267, 46,299, and 50,695 in group B, which may reflect their slightly different genetic traits. These differences were detected in all plasmids except for two, pCP-O13-19-1 and pCP-A19-1-1 (Table 4), as the sequences of these two plasmids were completely identical, indicating that strains O13-19 and A19-1 acquired their plasmids via recent HGT events or vertical inheritance from a common ancestor, although these strains were isolated from temporally and geographically different locations (June 2013 in Osaka and January 2019 in Aichi Prefecture, Japan, respectively) and from different sources, that is, a patient with gastroenteritis and oysters (Table S1).
To further determine whether the bec-encoding plasmids were acquired by vertical or horizontal gene transfer, we compared the accumulation of SNPs among all possible pairs of bec-encoding plasmids and cognate chromosomes of the bec-harbouring C. Fig. 2 Maximum likelihood phylogenetic tree of 216 C. perfringens strains. According to their phylogenetic relationships, the genomes were classified into five clades. The presence and absence of typical C. perfringens toxin genes, such as chromosomal plc, colA, pfoA, cpb2, and cpe (chromosomal or plasmid-borne), and plasmid-borne netB, alv, netF, tpeL, cpb, etx, becA, becB, iap, ibp, and lam (encoding the thermolysin-like metalloprotease called lambda toxin) [30], are indicated as different colour cells: black for the presence of chromosomal toxin genes, grey for the presence of plasmid-borne toxin genes, and light grey for the absence of the toxin gene. The toxinotyping of C. perfringens was determined based on the classification scheme proposed by Rood and co-workers and is indicated with seven colours [10]. In this study, the strains harbouring netB and cpe were classified as type G. The presence of the Pcp locus is shown as a red cell. Strain 13, which has pCP13, a representative plasmid of the pCP13-like family, is highlighted in blue, and the six bec-harbouring strains are highlighted in green perfringens strains (Tables 5, 6 and S5). In contrast to draft or whole genomes, complete genome information allows us to calculate the numbers and accumulation rates of SNPs for the plasmids and chromosomes separately, even if a strain contains more than one type of plasmid.
The chromosomal pair-wise comparisons of SNPs showed the largest number between strain OS1 and the other strains (64,905-66,800 per 2,710,984 bp, accounting for 2.39-2.46% of the aligned sequences) among all examined pairs (Tables 5 and S5-1), which is consistent with the phylogenetic tree, in which strain OS1 was    Fig. 2). These accumulation rates were much higher than those of the plasmid pair-wise SNPs (0.00551-0.0349%; Tables 6 and S5), which supported an inter-clade HGT event between strain OS1 and the other bec-harbouring strains. The number of chromosomal pair-wise SNPs detected in the comparisons among the other five strains (except between strains TS1 and A18-256), which were classified into clade 3, were approximately half (28,922-33,047, accounting for 1.07-1.22% of the aligned sequence) of the numbers for strain OS1 (Tables 5 and S5-1). These accumulation rates were also much higher than those of the plasmid pair-wise SNPs (0-0.0312%; Tables 6  and S5). The number of chromosomal pair-wise SNPs between strains TS1 and A18-256 was remarkably small (52, accounting for 0.00192% of the aligned sequence; Tables 5 and S5-1), which strongly suggested that these two strains, isolated from two distinct outbreaks, originated from nearly clonal strains. The accumulation rates of SNPs were statistically analysed. The rate of plasmid pair-wise SNPs between strains TS1 and A18-256 was not notably different from that of the chromosomal SNPs (p = 1, z = −0.04), which indicates that the bec-encoding plasmid was vertically inherited between these two strains. In contrast, the accumulation rates of the other plasmid pair-wise SNPs were much lower than those of the chromosomes (p < 2.2e-16, −37.01 ≤ z ≤ −24.19; Table S6). Consequently, these statistical results further supported the conclusion that the bec-encoding plasmids were inherited not only via inter-clade but also via intra-clade HGT.

Distribution of pCP13-like family plasmids
Our comparative analysis of complete genome information demonstrated that bec-encoding plasmids carry a conjugative Pcp locus, which is characteristic of pCP13-like family plasmids. The pCW3-like family is another family of conjugative C. perfringens plasmids that carry the tcp locus, and these plasmids are known to be disseminated among C. perfringens clades [20,34]. In contrast, the distribution of pCP13-like family plasmids has not been investigated, and only a limited number of pCP13-like family member plasmids, including pCP13 from strain 13, pCPNY83906550-1 from strain NY83906550, pCPT1 from strain T1, and p1 from strain JXJA17 (Table S7) have been previously known [22,24,27,35].
Thus, we examined the 216 assembled C. perfringens genomes, including our five bec-harbouring strains, to explore the distribution of pCP13-like family plasmids and found 58 strains (26.85%) likely had a Pcp locus (Fig. 2). Fifty-seven of the strains (the exception being strain NCTC8797, which lacks several parts of the Pcp locus) were confirmed to have both pcpB4 and pcpD4 genes, which encode a conjugation-specific VirB4-like ATPase and a VirD4-like coupling protein, respectively, which are crucial for conjugative transfer [22,36]. The plasmids are distributed broadly across several C. perfringens clades (clades 3, 4 and 5), suggesting that a wide variety of C. perfringens strains can acquire pCP13-like family plasmids via HGT events.

Comparative genomics of pCP13-like family plasmids
To compare the gene structure and organisation of the Pcp locus and accessory regions of the pCP13-like family plasmids, we selected 11 plasmids as representatives from various C. perfringens clades that contain Pcp locusharbouring strains based on their sequence alignment (Fig. S3). The results showed that the 11 plasmids shared a highly conserved sequence in the Pcp locus (>92% nt identity) (Fig. 3). Although the conjugation of pCP13-like family plasmids has recently been experimentally shown between two strain 13 derivatives, in future studies, it might be interesting to perform additional conjugation experiments using the strains described above to examine inter-clade transfer of pCP13-like family plasmids.
In the accessory regions, which encode several recombinase genes, the sequence lengths and numbers of encoded genes varied depending on the plasmid (Fig. 3 and Table 7), indicating that this region can incorporate a wide variety of foreign genes, as is also the case for the becA/becB and cpb2 genes. Importantly, numerous genes encoded in these regions are still functionally uncharacterized and are currently annotated as 'hypothetical'  (Table 7). Therefore, further characterisation and monitoring of the genes in this plasmid family are required. Since pCP13-like family plasmids have long been unrecognised, to the best of our knowledge, no detection methods that efficiently identify such family plasmids have been developed. In this study, our comparative genomics revealed extremely high sequence conservation of the Pcp locus among many pCP13-like family plasmids. This result may afford the design of an efficient, cost-effective PCR-based detection system that is helpful for monitoring and determining the virulence potential of pCP13-like family plasmids distributed worldwide.

Conclusion
In the present study, we reported five complete genomes of bec-harbouring C. perfringens strains isolated in 2009-2019. Analysis of the genomes showed that all the bec-encoding plasmids were pCP13-like plasmids and were disseminated among C. perfringens strains via HGT events, except for strains TS1 and A18-256, which appeared to be clonal. A previous study demonstrated the high transfer efficiency of the conjugative pCP13 plasmid in vitro [22], and our comparative genomics and data mining approach suggested that pCP13 and other plasmids in this family encoding conjugative Pcp loci were widely disseminated among C. perfringens strains (58/216 strains) in the natural environment. Additionally, the accessory regions of this plasmid family were potentially capable of integrating a wide variety of foreign genes, including virulence genes, such as the bec gene. These results demonstrate that care must be taken to monitor this newly characterised virulence plasmid in the surveillance of pathogenic C. perfringens.

Bacterial strains and genomic DNA extraction
The five bec-harbouring C. perfringens strains (OS1, TS1, O13-19, A18-256, and A19-1) sequenced in this study were isolated in Japan. OS1, TS1 [17], and A18-256 were isolated from faecal specimens of patients during distinct C. perfringens foodborne outbreaks in 2009, 2010, and 2018, respectively; O13-19 was isolated from a faecal specimen of a patient during a non-C. perfringens foodborne outbreak in 2013; and A19-1 was isolated from an oyster in 2019. All strains were anaerobically cultured in GAM broth (Nissui) at 37 °C, and then harvested by centrifugation at 3000 rpm for 20 min and subsequent collection of the cell pellet. Genomic DNA was extracted from the resuspended cells using the DNeasy PowerSoil Kit (QIAGEN).

Genome sequencing and assembly
The extracted genomic DNA of the five BEC-producing C. perfringens strains was sequenced using short and long sequencing platforms. The genomic DNA from strains OS1 and TS1 was sequenced using the PacBio RS II platform (Pacific Biosciences) and the SMRTbell Template Preparation Kit 1.0 (Pacific Biosciences). The genomic DNA from strains O13-19, A18-256, and A19-1 was sequenced using MinION (Oxford Nanopore Technologies) and the SQK-LSK109 1D ligation library preparation kit (Oxford Nanopore Technologies). MiSeq (Illumina) sequencing was also performed for the five strains using a paired-end strategy (2 × 150 cycles or 2 × 250 cycles) with the KAPA Hyper Plus library preparation kit (KAPA Biosystems). The sequencing reads produced by the PacBio RS II platform were assembled using HGAP v3 software [37]. The partial sequencing reads (a total of 3.3 Mbp × 50 lengths of sequencing reads) produced by MinION were assembled using Flye v2.5 software [38]. Assemblies with reads from MinION were corrected using Minimap2 v2.17 [39], and the assembled sequences of the five strains using reads from MiSeq were corrected using Pilon v1.23 [40].

Sequence determination of six bec-encoding plasmids
The sequences of the five assembled bec-encoding plasmids were polished using MiSeq reads and Pilon v1.23. The polished sequences were aligned using MAFFT v7.475 and manually checked, followed by Sanger sequencing of any sequences with putative errors.
The raw sequencing reads of Q135.2 were retrieved from the European Nucleotide Archive (ENA) (project PRJEB33762). The reads were quality trimmed and adapter removed using TrimGalore v0.6.4, with default options, followed by de novo assembly using Unicycler v 0.4.8 [41].

Genome annotation and calculating the ANI and dDDH
All assembled sequences were annotated using Prokka v1.14.5 [42]. The sequence of pCP-OS1 was manually annotated using pCP13 as a reference. The sequences encoding the dnaA gene were set as the chromosomal sequences. For all assemblies, the average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) were calculated using fastANI v1.3 [43] and GGDC v2.1 [44], respectively, which confirmed that all ANI and dDDH values were > 95 and > 70%, respectively.

Phylogenetic analysis of the core genome
A total of 211 assembled genomes of C. perfringens were downloaded from GenBank. Phylogenetic trees of 216 genomes, including our five strains, were constructed by the maximum likelihood method using Parsnp v1.2, with strain 13 as the reference genome, using the -x option to remove potential recent recombinations [45]. To determine the genetic distance, a VCF file of the tree was generated using Gingr [45]. The phylogenetic tree was visualised using the R packages phylogram, ggtree [46], ape, and RColorBrewer along with the presence of toxin genes and strain toxinotypes. Population structure was analysed using RhierBAPS with default parameters to assign the 216 strains to phylogenetic clades [32].

Comparing SNP accumulation between chromosomes and bec-encoding plasmids
The chromosomal sequences of the five newly sequenced strains, OS1, TS1, O13-19, A18-256, and A19-1, and the whole genome sequence of Q135.2 were aligned using Parsnp v1.2. Based on the generated VCF file, the lengths of the common sequences and the numbers of SNPs were determined. The six bec-encoding plasmids were also analysed in the same way as the chromosomes, that is, aligned using MAFFT software v7.475, followed by manual confirmation of SNPs and indels. The aligned common sequences were considered to be the core genomes of these plasmids. The SNP accumulation rates were calculated as the number of SNPs divided by the length of the common sequences, that is, 2,710,984 bp for the chromosomes, and 54,466 bp for the bec-encoding plasmids. Binomial tests were conducted for pairwise accumulation of SNPs between chromosomes and plasmids using R version 4.2.0, based on the rates of each chromosome pair.

Exploring the pCP13-like plasmids in public NGS data
We explored the Pcp locus in the 211 assembled genomes from GenBank using BLASTn using the Pcp locus sequence of pCP13 as a query. Candidate contigs encoding pcpB4 and pcpD4 were examined. The strains possessing these genes were identified as probable pCP13-like family plasmid carriers.